Synthesis of Novel C/D Ring Modified Bile Acids

Bile acid receptors have been identified as important targets for the development of new therapeutics to treat various metabolic and inflammatory diseases. The synthesis of new bile acid analogues can help elucidate structure–activity relationships and define compounds that activate these receptors selectively. Towards this, access to large quantities of a chenodeoxycholic acid derivative bearing a C-12 methyl and a C-13 to C-14 double bond provided an interesting scaffold to investigate the chemical manipulation of the C/D ring junction in bile acids. The reactivity of this alkene substrate with various zinc carbenoid species showed that those generated using the Furukawa methodology achieved selective α-cyclopropanation, whereas those generated using the Shi methodology reacted in an unexpected manner giving rise to a rearranged skeleton whereby the C ring has undergone contraction to form a novel spiro–furan ring system. Further derivatization of the cyclopropanated steroid included O-7 oxidation and epimerization to afford new bile acid derivatives for biological evaluation.


Introduction
Bile acids are amphiphilic compounds synthesized from cholesterol in the liver. These steroidal molecules possess both hydrophobic and hydrophilic regions enabling them to form micelles and function as physiological detergents for the absorption, distribution, metabolism and excretion of nutrients [1]. Bile acids also act as signaling molecules that activate the host cell receptors farnesoid X receptor (FXR) [2,3] and Takeda G proteincoupled receptor (GPCR) 5 [4,5] (TGR5; also known as G protein-coupled bile acid receptor 1, GPBAR1). FXR, a member of the nuclear receptor superfamily, is expressed at high levels in the liver and intestine and plays a central role in bile acid homeostasis alongside regulating various aspects of lipid [6,7] and glucose metabolism [7][8][9] as well as being involved in anti-inflammatory [10] and anti-fibrotic [11] activities. TGR5 belongs to the class A GPCR subfamily and is ubiquitously expressed in many tissues. Activation of TGR5 results in increased energy expenditure in adipose tissue and the secretion of glucagon-like peptide 1 (GLP-1), which is implicated in glucose metabolism and insulin sensitivity [12]. Consequently, dual FXR/TGR5 agonists were considered potential therapeutics for the treatment of metabolic disorders such as hypercholesterolemia, hypertriglyceridemia and type 2 diabetes. Concomitant activation of TGR5, however, presents with adverse effects such as pruritus [13], cholesterol gallstone formation [14] and gallbladder overfilling [15]. To avoid such side-effects, the development of new therapeutics to treat these diseases hinge on the ability to target these receptors selectively. Our approach towards achieving this was to synthesize a library of compounds with a modified C/D ring junction to be screened against these receptors to help elucidate key structure-activity relationships. junction to be screened against these receptors to help elucidate key structure-activity relationships.
As part of a program to develop new bile acids with targeted activities, we established a scalable route to the two novel alkenes 1 and 2 ( Figure 1) from cholic acid via a Nametkin-type rearrangement [16,17]. Of the various chemical manipulations afforded to olefins, we were particularly interested in applying those that result in the formation of new ring systems. Functionalizing the bile acids in this manner would not only afford a new class of compound to probe drug-receptor interactions but also advance investigations into the chemical reactivity of tetrasubstituted double bonds within the steroidal superstructure. Of the two chenodeoxycholic acid (CDCA, 3) derivatives isolated, the Δ 13 (14) -scaffold 1 provided a unique opportunity to investigate the cycloaddition of methylene across the C/D ring junction to generate bile acid propellanes with altered lipophilicity and structural conformations from the parent compound. To the best of our knowledge, there are no published examples of bile acid propellanes. The only known androstane-derived propellanes are the β-facing cyclopropanes 4-6 ( Figure 2), which were generated from deamination and a stereospecific rearrangement of 5α-H,18β-aminomethyl precursors [18][19][20]. Apart from a diastereomeric mixture of bile acids containing a side-chain cyclopropyl group across C-22 and C-23 (7) [21] and a spiro-cyclopropanyl derivative of obeticholic acid (8) [22], the only reported example of a bile acid containing a cyclopropyl at any position across the steroidal skeleton is compound 9 [22] (Figure 2).  Cyclopropanes, which can be formed with high diastereo-and enantiocontrol (as reviewed in [23,24]), have gained much attention in the fields of organic synthesis, medicinal chemistry and materials science for their interesting and unique properties [25]. The cyclopropanation of olefins can be catalyzed by various transition metal catalysts including zinc, copper, gold, palladium, and rhodium with others, such as ruthenium, iron, nickel, cobalt, titanium and yttrium, also having roles in cyclopropane formation [26]. Of  junction to be screened against these receptors to help elucidate key structure-activity relationships.
As part of a program to develop new bile acids with targeted activities, we established a scalable route to the two novel alkenes 1 and 2 ( Figure 1) from cholic acid via a Nametkin-type rearrangement [16,17]. Of the various chemical manipulations afforded to olefins, we were particularly interested in applying those that result in the formation of new ring systems. Functionalizing the bile acids in this manner would not only afford a new class of compound to probe drug-receptor interactions but also advance investigations into the chemical reactivity of tetrasubstituted double bonds within the steroidal superstructure. Of the two chenodeoxycholic acid (CDCA, 3) derivatives isolated, the Δ 13(14) -scaffold 1 provided a unique opportunity to investigate the cycloaddition of methylene across the C/D ring junction to generate bile acid propellanes with altered lipophilicity and structural conformations from the parent compound. To the best of our knowledge, there are no published examples of bile acid propellanes. The only known androstane-derived propellanes are the β-facing cyclopropanes 4-6 ( Figure 2), which were generated from deamination and a stereospecific rearrangement of 5α-H,18β-aminomethyl precursors [18][19][20]. Apart from a diastereomeric mixture of bile acids containing a side-chain cyclopropyl group across C-22 and C-23 (7) [21] and a spiro-cyclopropanyl derivative of obeticholic acid (8) [22], the only reported example of a bile acid containing a cyclopropyl at any position across the steroidal skeleton is compound 9 [22] (Figure 2).  Cyclopropanes, which can be formed with high diastereo-and enantiocontrol (as reviewed in [23,24]), have gained much attention in the fields of organic synthesis, medicinal chemistry and materials science for their interesting and unique properties [25]. The cyclopropanation of olefins can be catalyzed by various transition metal catalysts including zinc, copper, gold, palladium, and rhodium with others, such as ruthenium, iron, nickel, cobalt, titanium and yttrium, also having roles in cyclopropane formation [26]. Of Cyclopropanes, which can be formed with high diastereo-and enantiocontrol (as reviewed in [23,24]), have gained much attention in the fields of organic synthesis, medicinal chemistry and materials science for their interesting and unique properties [25]. The cyclopropanation of olefins can be catalyzed by various transition metal catalysts including zinc, copper, gold, palladium, and rhodium with others, such as ruthenium, iron, nickel, cobalt, titanium and yttrium, also having roles in cyclopropane formation [26]. Of these, zinc reagents often offer an inexpensive and less toxic approach to perform this functionalization.
Founded on the seminal work by Emschwiller [27], who in 1929 showed diiodomethane and zinc react to form an iodomethylzinc species, and Simmons and Smith [28,29] (for a review of the Simmons−Smith cyclopropanation, see [30]) who later reported the formal cycloaddition of methylene across various olefins by treatment of diiodomethane with the zinc−copper couple (for a review on the uses of zinc carbenoids in stereoselective synthesis, see [31]); new organozinc carbenoids have been successfully employed to cyclopropanate traditionally unreactive alkenes, expanding the scope of suitable olefinic substrates. Among these are the methods developed by the groups of Furukawa [32,33], Wittig and Denmark [34], Shi [35,36] and Charette [37].
Beyond the reactivity of the carbenoid species, the olefin substrate can profoundly influence cyclopropane stereoselectivity and product yield. Although electron-rich tetrasubstituted olefins can be favored through electronic factors, steric constraints can hinder reaction yields. To overcome such limitations, directing groups (i.e., those which contain Lewis-basic heteroatoms) can be used to chelate metal carbenoids positioning them within proximity of the olefin in order to execute the ring-forming step. In structure 1, we envisioned the homo-allylic alcohol at O-7 may provide such a handle.

Results and Discussion
In accordance with published procedures [16], our synthetic efforts began with cholic acid (10), which was suitably protected to allow selective mesylation at C-12 to give 13 (Scheme 1). Exposure to a sodium acetate/acetic acid buffer at 100 • C afforded the isomeric ∆ 13(14) -and ∆ 13(17) -alkenes 14 and 15 in a ratio of 1:2 as the major products. Hydrolysis of the methyl ester and acetate protecting groups followed by fractional crystallization from hot ethyl acetate/methanol mixtures afforded multi-gram quantities of 1 (7% from 13) and 2 (13% from 13) for further derivatization. these, zinc reagents often offer an inexpensive and less toxic approach to perform this functionalization. Founded on the seminal work by Emschwiller [27], who in 1929 showed diiodomethane and zinc react to form an iodomethylzinc species, and Simmons and Smith [28,29] (for a review of the Simmons−Smith cyclopropanation, see [30]) who later reported the formal cycloaddition of methylene across various olefins by treatment of diiodomethane with the zinc−copper couple (for a review on the uses of zinc carbenoids in stereoselective synthesis, see [31]); new organozinc carbenoids have been successfully employed to cyclopropanate traditionally unreactive alkenes, expanding the scope of suitable olefinic substrates. Among these are the methods developed by the groups of Furukawa [32,33], Wittig and Denmark [34], Shi [35,36] and Charette [37]. Beyond the reactivity of the carbenoid species, the olefin substrate can profoundly influence cyclopropane stereoselectivity and product yield. Although electron-rich tetrasubstituted olefins can be favored through electronic factors, steric constraints can hinder reaction yields. To overcome such limitations, directing groups (i.e., those which contain Lewis-basic heteroatoms) can be used to chelate metal carbenoids positioning them within proximity of the olefin in order to execute the ring-forming step. In structure 1, we envisioned the homo-allylic alcohol at O-7 may provide such a handle.
With the rearranged precursor bile acids 1 and 2 in hand, we turned our attention towards cyclopropane formation with the aim of using Zn-carbenoids to effect this transformation. To avoid unwanted Zn-chelation via the carboxylic acid, 1 was first treated with diazomethane to afford the corresponding methyl ester 16 in excellent yield (Scheme 2). The first attempt to cyclopropanate alkene 16 employed the Furukawa methodology whereby the corresponding Zn-carbenoid was formed in situ by reaction of Et2Zn and diiodomethane. Employing toluene as the solvent (80 °C, 42 h) afforded cyclopropane 17 exclusively in 73% yield; however, performing the same reaction in refluxing dichloromethane gave a similar quantity of product after only 4 h (Scheme 2). Given that Furukawa carbenoids are renowned for their stereospecificity and high reactivity with electron-rich olefins, it was not surprising that a high yield of a single cyclopropane diastereomer was obtained from this reaction. Although the facial orientation of the cyclopropane moiety of 17 could not be established by NMR spectroscopy, it was anticipated that the With the rearranged precursor bile acids 1 and 2 in hand, we turned our attention towards cyclopropane formation with the aim of using Zn-carbenoids to effect this transformation. To avoid unwanted Zn-chelation via the carboxylic acid, 1 was first treated with diazomethane to afford the corresponding methyl ester 16 in excellent yield (Scheme 2). The first attempt to cyclopropanate alkene 16 employed the Furukawa methodology whereby the corresponding Zn-carbenoid was formed in situ by reaction of Et 2 Zn and diiodomethane. Employing toluene as the solvent (80 • C, 42 h) afforded cyclopropane 17 exclusively in 73% yield; however, performing the same reaction in refluxing dichloromethane gave a similar quantity of product after only 4 h (Scheme 2). Given that Furukawa carbenoids are renowned for their stereospecificity and high reactivity with electron-rich olefins, it was not surprising that a high yield of a single cyclopropane diastereomer was obtained from this reaction. Although the facial orientation of the cyclopropane moiety of 17 could not be established by NMR spectroscopy, it was anticipated that the homo-allylic 7-OH group would direct cyclopropanation via the α-face. Hydrolysis of ester 17 produced carboxylic acid 18, and suitable crystals were obtained for X-ray diffraction, which un-equivocally proved the stereochemistry of the newly installed cyclopropane to be cis to the 7-OH as anticipated. This is the first reported example of an α-facing cyclopropane across the C/D ring junction of any androstane-containing motif. Because bile acid conjugates, typically as taurine and glycine salts, are more readily transported in biological systems, converting 18 into its taurine analogue was considered prudent for biological evaluation. This was achieved by first forming the carbonic anhydride of 18 in situ before reaction with taurine to afford conjugate 19. homo-allylic 7-OH group would direct cyclopropanation via the α-face. Hydrolysis of ester 17 produced carboxylic acid 18, and suitable crystals were obtained for X-ray diffraction, which unequivocally proved the stereochemistry of the newly installed cyclopropane to be cis to the 7-OH as anticipated. This is the first reported example of an α-facing cyclopropane across the C/D ring junction of any androstane-containing motif. Because bile acid conjugates, typically as taurine and glycine salts, are more readily transported in biological systems, converting 18 into its taurine analogue was considered prudent for biological evaluation. This was achieved by first forming the carbonic anhydride of 18 in situ before reaction with taurine to afford conjugate 19. The orientation of the 7-OH functionality in bile acids can have a pronounced effect on its biological activity. Compared to its 7α-epimer, CDCA, the 7β-hydroxy group of ursodeoxycholic acid (UDCA) renders this bile acid much more hydrophilic and therefore less toxic to gut bacteria [38]. However, CDCA has been shown to fully activate FXR, whereas UDCA had negligible activity on this receptor [39]. To investigate how the 7-OH functionality in 18 can influence potential receptor-agonist activities, a small library of bile acid propellanes was sought for biological testing. For this purpose, inversion of the C-7 hydroxyl group was attempted via oxidation and stereoselective reduction in the ketone moiety.
A report [40] describing the selective oxidation of the 7-hydroxyl of CDCA, in the presence of an unprotected 3-hydroxyl group, with pyridinium chlorochromate (PCC) have attributed its regioselectivity to the fact that the oxidation of axial hydroxyl groups over equatorial hydroxyl groups is kinetically favored. Reaction times exceeding 15 min, however, result in formation of the di-keto product [40]. In our hands, applying the reported conditions to diol 18 resulted in formation of the 3,7-di-keto product while significant amounts of starting material remained in solution. To avoid formation of undesired oxidation products, the 3-OH was instead temporarily protected as a benzoyl ester (16→20) before subjection to the Furukawa carbenoid methodology, affording the cyclopropane intermediate 21 in 71% yield over the two steps (Scheme 3). Alternatively, intermediate 21 was furnished in a similar overall yield (73%) from direct selective mono-benzolyation of cyclopropane-diol 17. With O-3 suitably protected and the cyclopropane installed, the oxidation of O-7 was screened using the common oxidants TEMPO/BAIB, NBS, KBr/NaOCl, PCC and Dess-Martin periodinane. From these, PCC proved to be the best oxidant for this substrate, affording the keto intermediate 22 in 95% yield, which was subsequently deprotected under basic reaction conditions to give 23. Based on reports that elemental sodium can be used to selectively reduce keto moieties on steroids to give βhydroxyl groups [41], 23 was treated with sodium in hot isopropanol. The desired 7β-OH The orientation of the 7-OH functionality in bile acids can have a pronounced effect on its biological activity. Compared to its 7α-epimer, CDCA, the 7β-hydroxy group of ursodeoxycholic acid (UDCA) renders this bile acid much more hydrophilic and therefore less toxic to gut bacteria [38]. However, CDCA has been shown to fully activate FXR, whereas UDCA had negligible activity on this receptor [39]. To investigate how the 7-OH functionality in 18 can influence potential receptor-agonist activities, a small library of bile acid propellanes was sought for biological testing. For this purpose, inversion of the C-7 hydroxyl group was attempted via oxidation and stereoselective reduction in the ketone moiety.
A report [40] describing the selective oxidation of the 7-hydroxyl of CDCA, in the presence of an unprotected 3-hydroxyl group, with pyridinium chlorochromate (PCC) have attributed its regioselectivity to the fact that the oxidation of axial hydroxyl groups over equatorial hydroxyl groups is kinetically favored. Reaction times exceeding 15 min, however, result in formation of the di-keto product [40]. In our hands, applying the reported conditions to diol 18 resulted in formation of the 3,7-di-keto product while significant amounts of starting material remained in solution. To avoid formation of undesired oxidation products, the 3-OH was instead temporarily protected as a benzoyl ester (16→20) before subjection to the Furukawa carbenoid methodology, affording the cyclopropane intermediate 21 in 71% yield over the two steps (Scheme 3). Alternatively, intermediate 21 was furnished in a similar overall yield (73%) from direct selective mono-benzolyation of cyclopropane-diol 17. With O-3 suitably protected and the cyclopropane installed, the oxidation of O-7 was screened using the common oxidants TEMPO/BAIB, NBS, KBr/NaOCl, PCC and Dess-Martin periodinane. From these, PCC proved to be the best oxidant for this substrate, affording the keto intermediate 22 in 95% yield, which was subsequently deprotected under basic reaction conditions to give 23. Based on reports that elemental sodium can be used to selectively reduce keto moieties on steroids to give β-hydroxyl groups [41], 23 was treated with sodium in hot isopropanol. The desired 7β-OH analogue 24 was formed as the major product, albeit in a modest 21% yield. The stereochemistry of the 7-epimers (18 and 24) was determined by X-ray analysis (e.g., compound 18) and by comparing the 1 H NMR spectra of compounds 18 and 24. For the 7α-OH-compound 18, H-7 eq typically appears as a quartet due to its coupling with adjacent protons with similar coupling constants (δ 4.11 (q, J = 2.9 Hz, 1H, H-7 eq )), whereas for 24, H-7 ax couples to H-6 ax and H-8 at ca. 180 • angles, and to H-6 eq at ca. 60 • , appearing as a triplet of doublets (δ 3.63 (td, J = 11.6, 4.8 Hz, 1H, H-7 ax )) and therefore distinguishable from its 7α-isomer.
analogue 24 was formed as the major product, albeit in a modest 21% yield. The stereochemistry of the 7-epimers (18 and 24) was determined by X-ray analysis (e.g., compound 18) and by comparing the 1 H NMR spectra of compounds 18 and 24. For the 7α-OH-compound 18, H-7eq typically appears as a quartet due to its coupling with adjacent protons with similar coupling constants (δ 4.11 (q, J = 2.9 Hz, 1H, H-7eq)), whereas for 24, H-7ax couples to H-6ax and H-8 at ca. 180° angles, and to H-6eq at ca. 60°, appearing as a triplet of doublets (δ 3.63 (td, J = 11.6, 4.8 Hz, 1H, H-7ax)) and therefore distinguishable from its 7αisomer. To investigate the importance of the proximity of the homo-allylic participating group at O-7 for cyclopropane formation, the Δ 13(17) -alkene isomer 25, with the alkene one carbon atom further removed from the directing group, was subjected to the Furukawa carbenoid for extended periods of time (Scheme 4). Monitoring the reaction by HPLC-MS showed only minor amounts (<10%) of putative cyclopropane formation as indicated by new peaks in the chromatogram with masses of M+14, with the bulk of the crude material comprised of starting material. The significance of the 7-hydroxyl group for directed carbene addition was further illustrated by subjecting the 3,7-di-OAc protected Δ 13(14) -alkene isomer (27) (the protected form of 16) to the same Furukawa conditions, but again, no significant amount of cyclopropane formation was observed. Notably, when protected alkene 27 was reacted with an excess of the Shi carbenoid, a reagent with higher reactivity than the Furukawa reagent towards isolated alkenes, no cyclopropane formation was detected. In an attempt to improve directed cyclopropanation reactions of the Δ 13(14) -substrate 16, alternative zinc-carbenoids were explored. Reacting 16 with the Simmons-Smith carbenoid (IZnCH2I, 4 equivalents) in refluxing diethyl ether and monitoring the reaction by LCMS failed to generate any significant new products with only starting material being detected. Interestingly, employing the Shi carbenoid (CF3C(O)OZnCH2I, 10 equivalents) in refluxing dichloromethane resulted in the formation of a novel spiro-derivative (29) in 7% yield alongside its 3-OMe analogue 30 as the major product (30% yield, based on 90% purity) contaminated with a small amount of an unidentified impurity (Scheme 5) that was difficult to remove by normal-phase chromatography. Methylation of the 3-hydroxyl in 30 was not entirely unexpected as the alkylation of heteroatoms is a known side product when using excess reagent and/or prolonged reaction times owing to the high electrophilicity of the zinc carbenoid [42]. Hydrolysis of methyl ester 29 gave its corresponding acid (31) for which X-ray crystal data were obtained, confirming formation of the new steroidal skeleton as depicted in Scheme 5.
To improve the yield of 29, it was proposed that protecting the 3-OH position of 16 would prevent formation of the methyl ether. Towards this, the selective protection of 3- In an attempt to improve directed cyclopropanation reactions of the ∆ 13(14) -substrate 16, alternative zinc-carbenoids were explored. Reacting 16 with the Simmons-Smith carbenoid (IZnCH 2 I, 4 equivalents) in refluxing diethyl ether and monitoring the reaction by LCMS failed to generate any significant new products with only starting material being detected. Interestingly, employing the Shi carbenoid (CF 3 C(O)OZnCH 2 I, 10 equivalents) in refluxing dichloromethane resulted in the formation of a novel spiro-derivative (29) in 7% yield alongside its 3-OMe analogue 30 as the major product (30% yield, based on 90% purity) contaminated with a small amount of an unidentified impurity (Scheme 5) that was difficult to remove by normal-phase chromatography. Methylation of the 3-hydroxyl in 30 was not entirely unexpected as the alkylation of heteroatoms is a known side product when using excess reagent and/or prolonged reaction times owing to the high electrophilicity of the zinc carbenoid [42]. Hydrolysis of methyl ester 29 gave its corresponding acid (31) for which X-ray crystal data were obtained, confirming formation of the new steroidal skeleton as depicted in Scheme 5. In an attempt to improve directed cyclopropanation reactions of the Δ 13(14) -substrate 16, alternative zinc-carbenoids were explored. Reacting 16 with the Simmons-Smith carbenoid (IZnCH2I, 4 equivalents) in refluxing diethyl ether and monitoring the reaction by LCMS failed to generate any significant new products with only starting material being detected. Interestingly, employing the Shi carbenoid (CF3C(O)OZnCH2I, 10 equivalents) in refluxing dichloromethane resulted in the formation of a novel spiro-derivative (29) in 7% yield alongside its 3-OMe analogue 30 as the major product (30% yield, based on 90% purity) contaminated with a small amount of an unidentified impurity (Scheme 5) that was difficult to remove by normal-phase chromatography. Methylation of the 3-hydroxyl in 30 was not entirely unexpected as the alkylation of heteroatoms is a known side product when using excess reagent and/or prolonged reaction times owing to the high electrophilicity of the zinc carbenoid [42]. Hydrolysis of methyl ester 29 gave its corresponding acid (31) for which X-ray crystal data were obtained, confirming formation of the new steroidal skeleton as depicted in Scheme 5.
To improve the yield of 29, it was proposed that protecting the 3-OH position of 16 would prevent formation of the methyl ether. Towards this, the selective protection of 3-OH was achieved with acetic anhydride and sodium bicarbonate in THF at 40 • C over 2 days. Increasing the reaction temperature to reflux gave a mixture of 7-OAc isomer and di-acetylated product after 2 h. With the 3-OAc compound (32) in hand, treatment with 10 equivalents of Shi's Zn-carbenoid afforded the spiro derivative 33 (51%, based on 90% purity) contaminated with small amounts of unidentified impurities that were difficult to remove by chromatography on silica gel (Scheme 6). Deprotection afforded 31 in 83% yield. OH was achieved with acetic anhydride and sodium bicarbonate in THF at 40 °C over 2 days. Increasing the reaction temperature to reflux gave a mixture of 7-OAc isomer and di-acetylated product after 2 h. With the 3-OAc compound (32) in hand, treatment with 10 equivalents of Shi's Zn-carbenoid afforded the spiro derivative 33 (51%, based on 90% purity) contaminated with small amounts of unidentified impurities that were difficult to remove by chromatography on silica gel (Scheme 6). Deprotection afforded 31 in 83% yield. Because propellanes have been noted to rearrange under acidic conditions [43] it was postulated that 29 could have been formed by trifluoracetic acid-promoted rearrangement of a cyclopropane intermediate leading to the more thermodynamically stable product isolated. To evaluate this possibility, cyclopropane 17 was treated with either TFA or acetic acid and monitored by NMR spectroscopy. However, after 5 min (TFA) or 4 h (AcOH), although the cyclopropane had been consumed, there was no evidence of 29 forming under these conditions. Instead, a potential mechanism leading to 29 is proposed to occur through coordination of the more electron deficient Shi carbenoid species to O-7, which then withdraws electron density from the (13,14)-double bond to form a carbocation at C-13 (Scheme 7). Concomitant migration of the (8,14)-σ-bond with the vacant p-orbital of the nascent carbocation at C-13, due to excellent orbital overlap, is followed by spiro-etherification of O-7 at the newly formed carbocation, then quenching of the resultant carbenoid species. This rearrangement process may be highly concerted based on the minimal atomic reorganization required, and to the best of our knowledge is an unprecedented and unexplored skeletal rearrangement in bile acid and steroid chemistry. Because propellanes have been noted to rearrange under acidic conditions [43] it was postulated that 29 could have been formed by trifluoracetic acid-promoted rearrangement of a cyclopropane intermediate leading to the more thermodynamically stable product isolated. To evaluate this possibility, cyclopropane 17 was treated with either TFA or acetic acid and monitored by NMR spectroscopy (Supplementary Materials). However, after 5 min (TFA) or 4 h (AcOH), although the cyclopropane had been consumed, there was no evidence of 29 forming under these conditions. Instead, a potential mechanism leading to 29 is proposed to occur through coordination of the more electron deficient Shi carbenoid species to O-7, which then withdraws electron density from the (13,14)-double bond to form a carbocation at C-13 (Scheme 7). Concomitant migration of the (8,14)-σ-bond with the vacant p-orbital of the nascent carbocation at C-13, due to excellent orbital overlap, is followed by spiro-etherification of O-7 at the newly formed carbocation, then quenching of the resultant carbenoid species. This rearrangement process may be highly concerted based on the minimal atomic reorganization required, and to the best of our knowledge is an unprecedented and unexplored skeletal rearrangement in bile acid and steroid chemistry. OH was achieved with acetic anhydride and sodium bicarbonate in THF at 40 °C over 2 days. Increasing the reaction temperature to reflux gave a mixture of 7-OAc isomer and di-acetylated product after 2 h. With the 3-OAc compound (32) in hand, treatment with 10 equivalents of Shi's Zn-carbenoid afforded the spiro derivative 33 (51%, based on 90% purity) contaminated with small amounts of unidentified impurities that were difficult to remove by chromatography on silica gel (Scheme 6). Deprotection afforded 31 in 83% yield. Because propellanes have been noted to rearrange under acidic conditions [43] it was postulated that 29 could have been formed by trifluoracetic acid-promoted rearrangement of a cyclopropane intermediate leading to the more thermodynamically stable product isolated. To evaluate this possibility, cyclopropane 17 was treated with either TFA or acetic acid and monitored by NMR spectroscopy. However, after 5 min (TFA) or 4 h (AcOH), although the cyclopropane had been consumed, there was no evidence of 29 forming under these conditions. Instead, a potential mechanism leading to 29 is proposed to occur through coordination of the more electron deficient Shi carbenoid species to O-7, which then withdraws electron density from the (13,14)-double bond to form a carbocation at C-13 (Scheme 7). Concomitant migration of the (8,14)-σ-bond with the vacant p-orbital of the nascent carbocation at C-13, due to excellent orbital overlap, is followed by spiro-etherification of O-7 at the newly formed carbocation, then quenching of the resultant carbenoid species. This rearrangement process may be highly concerted based on the minimal atomic reorganization required, and to the best of our knowledge is an unprecedented and unexplored skeletal rearrangement in bile acid and steroid chemistry.

Conclusions
The stereoselective α-cyclopropanation of a ∆ 13(14) -bile acid alkene (16 or its 3-OHprotected form 20), in which the 7-OH is not protected, occurs readily when it is treated with the Furukawa carbenoid via assisted intramolecular addition. Applying the same conditions to either the isomeric ∆ 13(17) -bile acid 2 or to compound 27, where the 7-OH is protected, abolishes cyclopropane formation showing the importance of the homo-allylic hydroxyl for directed methylene cycloaddition in this system. The cyclopropane product was further derivatized as the taurine conjugate and converted to a 7-keto-derivative and its 7β-epimer. In contrast to treatment with the Furukawa carbenoid, when the ∆ 13(14) -alkene (16 or its 3-OH-protected form 32) was exposed to the Shi carbenoid, the major product was an unexpected rearranged spiro-furan derivative, the structure of which was elucidated through X-ray crystallography. Overall, this study has resulted in bile acid analogues with altered C/D ring conformations, which are currently being investigated for their ability to selectively activate the bile acid receptors FXR and TGR5.

Materials and Methods
Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded on Bruker Avance (III)-500 MHz spectrometer. Chemical shifts are reported in ppm relative to Me 4 Si (TMS, δ 0.00 ppm), or residual solvent peaks as an internal standard set to δ 7.26 and 77.16 ppm (CDCl 3 ), or δ 3.31 and 49.00 ppm (CD 3 OD), or δ 2.50 and 39.52 ppm (d 6 -DMSO) or δ 7.01 and 20.43 ppm (toluene-d 8 ). Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed on a Waters QTOF Premier mass spectrometer (Micromass, UK) under normal conditions. Sodium formate solution was used as calibrant for high resolution mass spectra (HRMS) measurements. Reactions were monitored by thin layer chromatography (TLC) using 0.2 mm silica gel (Merck Kieselgel 60 F254) precoated aluminum plates, using UV light, ammonium molybdate or potassium permanganate staining solution to visualize. Silver nitrate-impregnated TLC plates were prepared by dipping silica gel precoated aluminum plates in a 20% silver nitrate solution in acetonitrile and drying these in an oven at 120 • C. Silver nitrate-impregnated silica gel was prepared by dispersing silica gel in a solution of the corresponding amount of silver nitrate in acetonitrile and concentrating this mixture to dryness. Flash column chromatography was performed on Davisil ® silica gel (60, particle size 40-63 µm), or using Reveleris ® silica or C18 reversed phase flash cartridges on a Grace Reveleris ® automated flash system with continuous gradient facility. Solvents for reactions and chromatography were analytical grade and were used as supplied unless otherwise stated. Crystal structures were collected on an Agilent SuperNova diffractometer fitted with an EOS S2 detector, using CuKα radiation (1.54184 Å) at 120 K.